Extraction, purification, identification and metabolism of 3′,5′-cyclic UMP, 3′,5′-cyclic IMP and 3′,5′-cyclic dTMP from rat tissues

Newton, Russell P.; Kingston, Eric E.; Hakeem, N. A.; Salih, Saad G.; Beynon, J. H.; Moyse, Christopher D.

doi:10.1042/bj2360431

Cited by 46 publications

(26 citation statements)

References 25 publications

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“…These data raise the intriguing question whether UTP and CTP can also be substrates for ACs. In support of such a possibility is the fact that both 3Ј,5Ј-cyclic CMP and 3Ј,5Ј-cyclic UMP do, indeed, occur in mammalian tissues as shown by sensitive mass spectrometry techniques (Newton et al, 1984(Newton et al, , 1986. Moreover, in experiments with [␣-32 P]UTP as substrate, [ 32 P]cUMP was identified as a bona fide product of sGC (Gille et al, 2004), but direct structural confirmation of cUMP production by sGC with mass spectrometry techniques is still missing.…”

Section: Discussionmentioning

confidence: 92%

Differential Inhibition of Various Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by 2′,3′-O-(2,4,6-Trinitrophenyl)-Substituted Nucleoside 5′-Triphosphates

Suryanarayana¹,

Göttle²,

Hübner³

et al. 2009

J Pharmacol Exp Ther

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Adenylyl cyclases (ACs) catalyze the conversion of ATP into the second messenger cAMP and play a key role in signal transduction. In a recent study (Mol Pharmacol 70: 878 -886, 2006), we reported that 2Ј,3Ј-O-(2,4,6-trinitrophenyl)-substituted nucleoside 5Ј-triphosphates (TNP-NTPs) are potent inhibitors (K i values in the 10 nM range) of the purified catalytic subunits VC1 and IIC2 of membranous AC (mAC). The crystal structure of VC1:IIC2 in complex with TNP-ATP revealed that the nucleotide binds to the catalytic site with the TNP-group projecting into a hydrophobic pocket. The aims of this study were to analyze the interaction of TNP-nucleotides with VC1:IIC2 by fluorescence spectroscopy and to analyze inhibition of mAC isoforms, soluble AC (sAC), soluble guanylyl cyclase (sGC), and G-proteins by TNP-nucleotides. Interaction of VC1:IIC2 with TNP-NDPs and TNP-NTPs resulted in large fluorescence increases that were differentially reduced by a water-soluble forskolin analog. TNP-ATP turned out to be the most potent inhibitor for ACV (K i , 3.7 nM) and sGC (K i , 7.3 nM). TNP-UTP was identified as the most potent inhibitor for ACI (K i , 7.1 nM) and ACII (K i , 24 nM). TNP-NTPs inhibited sAC and GTP hydrolysis by G s -and G iproteins only with low potencies. Molecular modeling revealed that TNP-GTP and TNP-ATP interact very similarly, but not identically, with VC1:IIC2. Collectively, our data show that TNPnucleotides are useful fluorescent probes to monitor conformational changes in VC1:IIC2 and that TNP-NTPs are a promising starting point to develop isoform-selective AC and sGC inhibitors. TNP-ATP is the most potent sGC inhibitor known so far.ACs catalyze the conversion of ATP into the second messenger cAMP and play a key role in signal transduction. In mammals, nine membranous AC isoforms (ACI-IX) have been identified (Defer et al., 2000;Sunahara and Taussig, 2002). mACs differ from each other in regulation and tissue expression, and studies with transgenic and gene-knock-out mice have revealed different functions for the various AC isoforms. Most strikingly, ACV is an important AC isoform in the heart (Göttle et al., 2009), and the knock-out of ACV protects mice against heart failure and induces longevity (Yan et al., 2007). Thus, ACV inhibitors may be valuable drugs for the treatment of cardiovascular diseases and ageing (Iwatsubo et al., 2004;Göttle et al., 2009). In addition to mACs, mammals express a structurally distinct sAC that is predominantly found in testis and is important for sperm maturation (Chen et al., 2000). Thus, sAC inhibitors may be promising candidates for male contraceptives (Schlicker et al., 2008). Finally, sGC, catalyzing the conversion of GTP into O-(2,4,6-trinitrophenyl); VC1 and IIC2, the N-and C-terminal catalytic domains from canine type V mAC and rat type II mAC, respectively, expressed as soluble proteins; GTP␥S, guanosine 5Ј-[␥-thio]triphosphate.

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Section: Discussionmentioning

confidence: 92%

Differential Inhibition of Various Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by 2′,3′-O-(2,4,6-Trinitrophenyl)-Substituted Nucleoside 5′-Triphosphates

Suryanarayana¹,

Göttle²,

Hübner³

et al. 2009

J Pharmacol Exp Ther

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“…Pyrimidine cyclic nucleotides were first identified in the 1960s to 1980s (10,11,18,23,41,42), with evidence for expression of enzymes that synthesize and degrade cUMP and/or cCMP (12,23,30,31). However, studies were confounded by limitations in the available research tools, including antibodies that were not sufficiently selective to discriminate between contaminating molecular species.…”

Section: Discussionmentioning

confidence: 99%

Heterogeneity of pulmonary endothelial cyclic nucleotide response toPseudomonas aeruginosaExoY infection

Morrow

Seifert

Kaever

et al. 2015

American Journal of Physiology-Lung Cellular and Molecular Phys

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“…Moreover, ATP and UTP were reported previously to inhibit sGC competitively (39 -41). Furthermore, cUMP occurs at low concentrations in mammalian tissues (77,78), but its enzymatic origin has remained unknown. The low cUMP levels conform to the low UC activity of sGC.…”

Section: Potent and Selective Ac Inhibition By Mant-itp␥s Relative Tomentioning

confidence: 99%

Differential Inhibition of Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by Purine and Pyrimidine Nucleotides

Gille

Lushington

Mou

et al. 2004

Journal of Biological Chemistry

215

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ACs 1 catalyze the conversion of ATP into the second messenger cAMP, PP i being the second product of the cyclase reaction. Mammals express nine membranous ACs (ACs 1-9) (1, 2) and a sAC that is predominantly expressed in testis (3). Bacillus anthracis and Bacillus pertussis produce the AC toxins EF and ACT, respectively, that are activated by Ca 2ϩ /calmodulin and act through excessive cAMP accumulation in host cells (4,5). sGC is structurally related to ACs 1-9 in the catalytic site and is activated by [6][7][8]. sGC catalyzes the formation of the second messenger cGMP from GTP. ACs 1-9 contain a tandem repeat structure with two transmembrane domains and two cytosolic domains (1, 2). The cytosolic domains are referred to as C1 and C2, respectively. Together, C1 and C2 form the catalytic site of AC. C1 and C2 also contain the regulatory sites for the stimulatory G-protein, G␣ s , for the inhibitory G-protein, G␣ i , and for the diterpene, forskolin. Catalytic activity of all AC isoforms depends on the presence of divalent cations (Mg 2ϩ or Mn 2ϩ ). Membranous ACs possess two Me 2ϩ -binding sites (9 -11). When mixed together, purified C1 and C2 form a functional AC that is efficiently activated by forskolin and G␣ s -GTP␥S (12, 13).AC isoforms differ from each other in their regulation (1, 2). ACs 1-9 are all activated by G␣ s , whereas sAC is activated by HCO 3 Ϫ (14). Forskolin activates ACs 1-8 but not AC9 or sAC. G␣ i inhibits ACs 1, 5, and 6. G-protein ␤␥ subunits exhibit stimulatory or inhibitory effects on AC isoforms. Ca 2ϩ /calmodulin stimulates ACs 1, 3, and 8. In addition, Mg 2ϩ and Mn 2ϩ show differential stimulatory effects on AC isoforms (15). More-

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Extraction, purification, identification and metabolism of 3′,5′-cyclic UMP, 3′,5′-cyclic IMP and 3′,5′-cyclic dTMP from rat tissues

Cited by 46 publications

References 25 publications

Differential Inhibition of Various Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by 2′,3′-O-(2,4,6-Trinitrophenyl)-Substituted Nucleoside 5′-Triphosphates

Differential Inhibition of Various Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by 2′,3′-O-(2,4,6-Trinitrophenyl)-Substituted Nucleoside 5′-Triphosphates

Heterogeneity of pulmonary endothelial cyclic nucleotide response toPseudomonas aeruginosaExoY infection

Differential Inhibition of Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by Purine and Pyrimidine Nucleotides

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